Previous studies suggest an innate sex difference in adenosine receptor density and/or function, which is mediated, at least in part, by NMDA receptors (Butler et al., 2008). The currently proposed studies will investigate effects of chronic ethanol (EtOH) exposure on adaptive changes in adenosine and NMDA receptor proteins, which may confer sensitivity to neurotoxicity during ethanol withdrawal (EWD) in a concentration- and/or sex-dependent manner. Male and female organotypic hippocampal slice cultures will be continuously exposed to EtOH (0, 25, or 50 mM) for ten days. Upon removal of EtOH, immunohistochemistry will be employed for the adenosine A1 and A2A receptor subtypes and the NMDA NR1 and NR2B subunits to quantify receptor density in the primary cell layers of the dentate gyrus, and the CA3 and CAI regions of the hippocampus. Western blotting will be conducted for an additional quantitative measure of changes in receptor proteins throughout the whole hippocampal formation in male and female EtOH-exposed cultures (0, 25, 50 mM). Additional studies will examine functional consequences of pharmacological antagonism of adenosine Al receptor antagonism during EWD. Cultures will be exposed to EtOH for ten days before twenty-four hour EWD. For the duration of EWD, cultures will be exposed to an Air antagonist;an Air antagonist + Air agonist;or an Air antagonist + NMDAr antagonist;and all cultures will be exposed to propidium iodide (PI) for labeling of dead/dying cells. Cultures will then be formalin-fixed for immunohistochemical labeling of mature neurons with neuronal nuclear protein (NeuN). Additional cultures will be exposed to the same drug treatments described, but will be exposed to Calcium (Ca2+) Orange at the end of twenty-four hour EWD to measure whether toxicity caused by A1r antagonism during EWD is correlated with potentiated Ca2+ influx. Slices will be visualized using fluorescent microscopy and data will be quantified using densitometry and confocal imaging. Correlational analyses will determine if the measures of cell viability (PI, NeuN, Ca2-*- orange) are related. These techniques allow for a comprehensive characterization of changes in the adenosinergic and NMDA receptor systems induced by EtOH exposure, and the functional consequences of those hypothesized adaptations during EWD. A significant amount of evidence suggests that the female brain of both humans and rodents is more sensitive to neurotoxicity in response to EtOH exposure and/or EWD. The current studies will contribute to our knowledge of EtOH- induced neuronal adaptations and potential sex differences in brain structure and/or function, and may contribute to the advent of novel pharmacotherapies to ameliorate EtOH and/or EWD-related toxicity.